Miniaturized head-mounted microscopes for in vivo recording of neural activity have gained much recognition within the past decade of neuroscience research. In combination with fluorescent reporters, these miniature microscopes allow researchers to record the neural activity that underlies behavior, cognition, and perception in freely moving animals. Single-photon miniature microscopes are convenient for widefield recording but lack the increased penetration depth and optical sectioning capabilities of multiphoton imaging. Here we discuss the current state of head-mounted multiphoton miniature microscopes and introduce a miniature head-mounted two-photon fiber-coupled microscope (2P-FCM) for neuronal imaging with active axial focusing enabled using a miniature electrowetting lens. The 2P-FCM enables three-dimensional two-photon optical recording of structure and activity at multiple focal planes in a freely moving mouse. Detailed methods are provided in this chapter on the 2P-FCM design, operation, and software for data analysis.
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